Somatic gene transfer offers a myriad of possibilities for therapeutic usage in a number of diseases including congenital defects, as well as acquired forms of pathological abnormalities. There have been several critical limitations that have hindered the practical application of gene transfer in vivo. These include the duration of expression of transferred genes, the trade-off between tissue specificity and the efficiency of gene expression, and the adverse side effects of local inflammation provoked by vectors.
In the field of cardiovascular medicine, gene therapy has been focused on vascular gene transfer, mainly aimed at ischemic coronary disease. Many research groups have attempted cardiac gene transfer using adenovirus (Ad) vectors with strong, non-tissue specific gene expression cassettes driven by cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. Clinical trials of several angiogenic factors including vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) have been ongoing. The expectation is that transduction of cardiac cells with viral vectors will result in the secretion of these growth factors from cardiac cells, inducing the growth of new blood vessels and improving the blood supply to the heart to decrease ischemia.
A few publications have reported some success in the modification of cardiac function through gene transfer experimentation in rats and rabbits using intra-aorta or intracoronary injection of virus. However, in these reports, either the extent or the specificity of gene transfer was not described properly, or the gene expression was patchy in distribution. Ascending aortic constriction was created in rats to stimulate compensatory hypertrophy which frequently results in heart failure (Miyamoto et al., 2000). Activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) is reduced in failing hearts, resulting in abnormal Ca2+ handling, and eventually leading to contractile failure. Intracardiac injection of an Ad-SERCA2a viral vector in rats was sufficient to induce some physiological improvement; however, there were a number of limitations. Ad vectors induced robust immunological response, resulting in myocardial necrosis. Such a robust immune response would prevent the re-administration of the Ad and may result in the clearance of the transduced cells by the immune system. Although the majority of the myocytes were claimed to express the transferred gene, there was no discussion in the report regarding the transduction of other cell types in the heart, or the tissue specificity of gene expression. In a similar study by the same investigators using normal rats, patchy expression of a reporter construct in the heart was reported. Additionally expression of the transferred gene in remote organs, including lung and liver was observed (Hajjar et al., 1998).
Other attempts to improve cardiac function have focused on the β2 adrenergic receptor (β-AR). Function of β-AR is decreased in heart failure and overexpression of β-AR in otherwise normal transgenic mice results in increased cardiac function. To determine if β-AR could increase cardiac function in normal rabbits, adenovirus expressing β-AR (Ad-β-AR) was injected into the left ventricle via catheter with the aorta cross clamped for 40 seconds (Maurice et al. 1999). The delivery method produced diffuse, multichamber myocardial expression, and cardiac function was improved; however, neither the efficiency nor the specificity of gene transfer was discussed in the report. Subsequently, β-adrenergic kinase inhibitor was delivered in the post myocardial infarction hearts of rabbits via an adenovirus vector to attenuate β-AR desensitization to achieve only regional improvement of cardiac contractility (Shah et al., 2001). Thus, the demonstration of the therapeutic effect of cardiac somatic gene transfer has been hampered by the lack of in vivo gene delivery strategies to effect long-term, high-efficiency, cardiotropic expression in the intact heart.
Dysfunction of one protein may be corrected by modulation of a regulatory factor of the protein. In a study by Minamisawa et al. (1999), it was demonstrated that deletion or disruption of phospholamban (PLB), a protein that inhibits the function of SERCA2, was able to rescue cardiac defects in a mouse in which the muscle-specific LIM protein (MLP) was deleted(Arber et al., 1997). A double-knock out (DKO) mouse was generated by crossing a PLB knockout mouse with a MLP knockout mouse. The resulting mice showed few of the defects of the MLP knockout mouse strain. In this regard, genetic complementation studies in a gene targeted mouse model of dilated cardiomyopathy have identified a pivotal role for defects in sarcoplasmic reticulum calcium cycling in heart failure progression (Chien, 1999).
Dominant negative mutants of a protein may be used to inhibit function of the protein, resulting essentially in a knock out mutation. A number of dominant negative mutants of PLB have been identified and characterized by various methods (WO 00/25804 incorporated herein by reference). These mutations include the point mutations E2A, K3E, R14E, S16N, S16E, L37A, I40A and V49A as well as the double mutation K3E/R14E, some of which have only been tested in vitro (Toyofuku et al, 1994). Neonatal injection into the ventricular cavity of an Ad viral vector expressing a dominant negative form of PLB (S16 E or V49A) inhibited function of the native PLB rescuing cardiac function. High efficiency long-term in vivo cardiotropic gene transfer in several forms of chronic heart failure model had been considered a critical test to evaluate the therapeutic value of functional modification of phospholamban.
It is known from studies on cystic fibrosis that transduction of all cells is not required for improved function. Expression of the wild type sodium channel in as few as 6-10% of cells within an epithelial sheet lacking a functional sodium channel is sufficient for normal sodium ion transport (Johnson et al, 1992). This is known as the bystander effect. It is likely that sporadic expression of a calcium channel or receptor may be sufficient to increase function of a diseased tissue; however, replacement of a structural protein would likely require more efficient gene transfer. To date, there are no reports of stable, high efficiency transfer of genes into cardiac tissue.
The naturally occurring autosomal recessive Syrain hamster cardiomyopathy (CM) in BIO14.6, UMX7.1 and TO-2 hamster strains have been identified recently as being due to a mutation in the δ-sarcoglycan gene (Nigro, et al. 1997; Sakamoto, et al., 1997). This mutation results in decreased stable expression of all of the sarcoglycan genes (α, β, and γ), resulting in decreased structural integrity in all muscle cells. The progressive left ventricular dilation, systolic and diastolic dysfunction, and cell loss in the CM hamster resemble many phenotypic features of human primary dilated cardiomyopathy (DCM) (Ryoke et al., 1999; Ikeda et al., 2000). In the hamster, these phenotypic changes are associated with increased myocardial cell permeability and rupture. In transgenic mice, disruption of the δ-sarcoglycan gene caused myocardial damage, reported to be associated at least partially with vascular smooth muscle abnormality (Coral-Vasquez et al., 1999). In transgenic mice, disruption of the δ-sarcoglycan gene has been reported to be associated with vascular smooth muscle abnormality and secondary myocardial damage (Coral-Vazquez, et al., 1999). The fragility of cells is believed to be due to the incorrect assembly of the dystrophin-associated glycoprotein complex (DAGC) (Sakamoto, et al., 1997). Normally the components of the DAGC exist perpendicular to the plane of the sarcolemma and bind to the extracellular matrix protein lanminin and the intracellular protein dystrophin, stabilizing the cell. In the absence of δ-sarcoglycan, the complex collapses and is no longer able to stabilize the cells, making them more susceptible to mechanical stress.
Rescue of skeletal muscle dystrophy of the CM hamster has been accomplished by intramuscular injection of Ad (Holt, et al., 1998) or adenovirus associated virus (AAV) (Greelish, et al., 1999) containing the δ-sarcoglycan gene. Rescue by Ad-δ-sarcoglycan was accomplished by direct injection of the Ad-δ-sarcoglycan into the quadriceps femoris. Expression was initially high (≧80%), and some expression was seen for up to 198 days post-viral administration; however, expression decreased significantly over time. There was no discussion of expression of the gene product, or lack thereof, at remote sites.
Direct injection of AAV-δ-sarcoglycan into a small muscle (i.e. tibialis anterior) was sufficient for gene delivery throughout the muscle; however, efficient gene delivery in larger muscles (i.e. hindlimb) required delivery via circulation with concurrent disruption of endothelium using histamine. The hindlimb was isolated from systemic circulation by tourniquets, followed by injection of papaverine in histamine, and finally virus into the femoral vessel. After 4 to 6 weeks, structural integrity of cells was tested in an ex vivo system. In both systems, treatment with the δ-sarcoglycan expressing virus resulted in increased structural stability of the muscle cells. There was no discussion of gene delivery to the heart. Additionally, the isolation of the heart from circulation by tourniquet to increase exposure of the virus to the tissue of interest would clearly be problematic; therefore, the same method could not be used for cardiac tissue.
In some cases of the hereditary dystrophies, such as Duchenne's muscular dystrophy, death is usually caused by heart failure due to cardiomyopathy rather than skeletal muscle myopathy, as is the case with CM in hamsters. As the sarcoglycan gene products are structural elements of the cell, efficient transfer to nearly all cells would be required for effective treatment of the disease. Therefore methods for efficient gene transfer into cardiac muscle would be useful in the treatment of a number of muscular dystrophies.
Efficient gene delivery to the heart presents a greater problem than delivery to striated muscle tissue due to structural differences of the tissues. Striated muscle cells are large, multinucleate cells that are derived from the fusion of multiple myoblasts. Therefore, delivery of a virus particle to a single cell would result in expression over a much larger area as the RNA transcribed in a single nucleus would be transported throughout the cell. Cardiac cells contain only one or two nuclei per cell and are much smaller (10-fold). Expression in the same percentage area would require the efficient transduction of a significantly higher number of cells.
Tissue specific promoters have been used to increase specificity of myocardial gene expression, but expression levels of the transferred gene were low (Rothmann et al., 1996). Another strategy to restrict expression of transferred genes to the heart has involved direct injection of the virus into the myocardium (Gutzman et al, 1993; French et al., 1994). Another attempt involved intrapericardial virus vector injection combined with proteinase treatment (Fromes et al., 1999). These manipulations achieved local gene delivery due to a lack of intense viral vector diffusion; however, the outcome of these methods is highly restricted gene expression with local tissue damage.
The efficiency of cardiomyocyte gene delivery by an AAV vector was documented in vitro using cultured rat neonatal cells, as well as in an ex vivo system using rat papillary muscle immersion (Maeda et al., 1998). Ex vivo AAV vector transfer followed by syngeneic heart transplantation was reported to achieve high efficiency marker gene expression (Svensson et al., 1999). Transcoronary delivery of AAV was attempted in porcine myocardium; however, extremely low gene transfer efficiency was observed (0.2%) (Kaplitt et al., 1996). To date, there is no report of high efficiency in vivo cardiotropic gene delivery system with long-term sustainable expression.
Development of efficient methods for gene transfer would likely allow the correction of a number of cardiac problems.